Complexes of double-stranded ribonucleic acid with polyquaternary amine

ABSTRACT

WHEREIN EACH OF A AND B, INDEPENDENT OF THE OTHER IS AN INTEGER OF FROM 2 TO 6 AND X IS A NUMBER WHICH IS SUCH THAT THE AVERAGE MOLECULAR WEIGHT OF THE POLYCATION DIVIDED BY THE EQUIVALENT WEIGHT WHICH IS THE MOLECULAR WEIGHT OF THE POLYCATION DIVIDED BY THE VALUE (2X + 2) IS NOT GREATER THAN 98, AND THE ANIONS ARE EITHER (A) DOUBLESTRANDED RIBONUCLEIC ACID POLYANIONS, SAID DOUBLE-STRANDED RIBOUCLEIC ACID BEING OF NATURAL ORIGIN, OR )B) POLYANIONS OF A DOUBLE-STRANDED RIBONUCLEIC ACID OF NATURAL ORIGIN WHICH HAS BEEN SUBJECT TO CHEMICAL OR ENZYMATIC REACTION WHICH ALTERS THE PRIMARY AND/OR SECONDARY AND/OR TERTIARY STRUCTURE, PROVIDED THAT THE RESULTANT RIBONUCLEIC ACID RETAINS A SUBSTANTIAL DEGREE OF BASE PAIRING BETWEEN COMPLEMENTARY STRANDS, SAID ANTIVIRAL COMPLEX HAVING MORE THAN 60% OF THE ANIONIC SITES ON THE DOUBLE-STRANDED RIBOUNCLIC ACID ANIONS NEUTRALIZED BY THE QUATERNARY CATIONIC SITES ON THE QUATERNARY POLYMER. 1. AN ANTIVIRAL COMPLEX WHICH IS A PRINCIPALLY IONIC COMPLEX SOLUBLE IN 0.15 M AQUEOUS SODIUM CHLORIDE SOLUTION IN WHICH THE CATIONS ARE ORGAIC POLYMER POLYCATION HAVING A PLURALITY OF QUATERNARY NITROGEN SITES LOCATED AT INTERVALS ALONG THE POLYMER CHAINS, SAID POLYCATIONS HAVING THE FORMULA   (H3C)2-N-(CH2)A-(N(+)(-CH3)2-(CH2)B-N(-CH3)2-(CH2)A)X-   N(-CH3)2

United States Patent O 3,845,033 COMPLEXES OF DOUBLE-STRANDED RIBONU- CLEIC ACID WITH POLYQUATERNARY AMINE Michael Raymond Harnden, Horsham, England, assignor to Beecham Group Limited, Brentford, England No Drawing. Filed Nov. 6, 1972, Ser. No. 303,955 Claims priority, application Great Britain, Nov. 5, 1971, 51,514/ 71 Int. Cl. C07d 51/50 US. Cl. 260-211.5 R 7 Claims ABSTRACT OF THE DISCLOSURE Complexes of polymeric polycations having a plurality of quaternary nitrogen sites and double-stranded ribonucleic acids of natural origin, or derivatives thereof, demonstrate antiviral activity.

' BACKGROUND OF THE INVENTION It is now generally recognized that double-stranded ribonucleic acids are potent inducers of interferons and thus should be of value in the broad spectrum prophylaxis of viral infections, and, to a lesser extent, in the treatment of such infections. Double-stranded ribonucleic acids of both natural and synthetic origin have been shown to possess interferon-inducing and antiviral activity in tissueculture and in whole animals. Among the specific sources of interferon-inducing double-stranded ribonucleic acid which have been reported are the virus particles found in some strains of Penicillium chrysogenum, P. funiculasum, P. stoloniferum, Aspergillus niger and A. foetidus; cytoplasmic polyhedrosis virus; reovirus 3 virion; and the replicative form of M82 coliphage and of MU9 mutant coliphage.

However, there is some evidence which suggests that double-stranded ribonucleic acid of natural origin may b'eu'nacceptably toxic in mammals, and its medical and veterinary use may be limited. There is thus a need for an antiviral agent which is less toxic than double-stranded ribonucleic acid alone, and which has comparable or better antiviral activity.

: Polybases such as spermine, spermidine, cadaverine,

2. F. Dianzani, S. Baron, C. E. Buckler, and H. B. Levy, Proc. Soc. Exptl. Biol. Med., 136, 1971, 1111.

3. J. Y. Richmond, Arch. fur die gesamate VirusfOrschung, 33, 1971, 242.

4. J. G. Tilles, Proc. Soc. Exptl. Med., 133(4), 1970, 1334.

5. A. Billiau, C. E. Bnckler, F. Dianzani, C. Uhlendorf, and S. Baron, Ann. N.Y. Acad. Sci., 173(1), 1970, 657. 6. B. D. Rosenquist, Am. J. Vet. Res., 32(1), 1971, 35.

'BRIEF SUMMARY OF THE INVENTION This invention is based on the discovery that polymeric polycations having a plurality of quaternary nitrogen sites form strong complexes with natural double-stranded ribonucleic acids, which complexes have good antiviral activity, at least in small mammals.

It should perhaps be noted that Gabbay et al. (Ann. N .Y. Acad. Sci. 171(3), 1970, 810; and Biochemistry, 10(9), 1971, 1665) have studied the interaction of some m0n0- meric quaternary ammonium compounds with (inter alia) the double-stranded synthetic ribonucleic acid Poly A:Po1y U. However, their reports concern only the physico-chemical properties and structure of the complexes, and in addition, they have not reported work with polyquaternary ammonium compounds. Also, British Patent No. 1,230,065, refers to the use of cetyltrimethylammonium bromide as a precipitating agent for double-stranded ribonucleic acid of natural origin. I have, however, tested the complex of cetyltrimethylammonium bromide with a natural doublestranded ribonucleic acid, and find no significant difference between the toxicity and antiviral activity of the complex, and that of the double-stranded ribonucleic acid itself.

DETAILED DESCRIPTION According to the present invention there is provided an antiviral substance which is a principally ionic complex in which the cations are organic polymer polycations which have a plurality of quaternary nitrogen sites located at intervals along the polymer chains and the anions are polylysine, protamine and diethylaminoethyl dextran have been used in the past to stabilize nucleic acids generally, (and some synthetic double-stranded ribonucleic acids in particular) against thermal denaturation and nuclease degradation. When it was discovered that synthetic double-stranded ribonucleic acids were inducers of inter feron, many workers believed that treatment of the double-stranded ribonucleic acid with polybases such as these would result in stabilization against degradation by ribonu clease, and thus in improved or prolonged in vivo antiviral activity. Most of the interferon-related biological work carried out in the past has involved the separate addition of the polybase to synthetic double-stranded ribonucleic acids, mainly Poly IzPoly C. However, the results of this work were not encouraging. Elevated interferon levels were obtained in tissue cultures (refs. 1, 2, 3, 4, 5 and 6 below) but where the activities of polybase-treated synthetic ribonucleic acids were examined in whole animals (refs. 1, 6) neither protection against virus nor toxicity were substantially changed.

REFERENCES either (a) double-stranded ribonucleic acid polyanions, said double-stranded ribonucleic acid being of natural origin or (b) polyanions of a double-stranded derivative of a double-stranded ribonucleic acid of natural origin.

The term double-stranded used in connection with ribonucleic acid refers to the characteristic whereby two ribonucleic acid molecules are associated by hydrogen bonding between complementary bases in each molecule. Ribonucleic acids may vary in the degrees of doublestrandedness.

The term double-stranded ribonucleic acid of natural origin means any double-stranded ribonucleic acid with is isolata'ble from a naturally-occurring source (e.g. those sources listed earlier in this specification), and excludes synthetic double-stranded ribonucleic acids such as Poly IzPoly C, Poly AzPoly U and Poly GzPoly C.

The term double-stranded derivative of a doublestranded ribonucleic acid of natural origin means any double-stranded ribonucleic acid of natural origin which has been subjected to a chemical or biochemical (e.g.

258 m while gradually raising the temperature of the material. The U.V. absorption value of a double-stranded material at this frequency increases with increasing temperature until a constant value is reached, corresponding to the absorption of the thermally denatured (i.e. singlestranded) ribonucleic acid. The diiference between the two extremes of absorption expressed as a percentage of the absorption of the double-stranded material is termed the hyperchromicity of that material.

When the U.V. absorption at 258 me of a doublestranded material is plotted against temperature, it is found that the absorption is greater at high than at low temperatures. The temperature at which the absorption is mid-way between the absorption of the double-stranded material and that of the thermally denatured (i.e. singlestranded) material is called the Tm of the material.

The cationic moiety present in the complexes of this invention has been defined as an organic polymer cation which has a plurality of quaternary nitrogen sites located at intervals along the polymer chain. One group of suitable organic polymer polycations is the group of structure (I):

wherein a and b are integers which are the same or different and each is from 2 to X is a number equal to or greater than 2 which depends on the length of the polymer chain.

Another group of suitable polycations is the group of formula (I) wherein the carbon chain between quaternary nitrogen sites contains olefinic or acetylenic bonds, or carries methyl substituents.

The polyanions present in the complex of this invention are (a) double-stranded ribonucleic acid polyanions, said double-stranded ribonucleic acid being of natural origin or (b) polyanions of a double-stranded derivative of a double-stranded ribonucleic acid of natural origin. Preferred sources of double-stranded ribonucleic acid include the virus-like particles found in certain of the Pen'icillia, e.g. P. chrysogenum (British Pat. No. 1,170,- 929), P. stoloniferum (Banks et al., Nature 218, 542 (1968), P. cyaneoyulvum (Banks et al., Nature 223, 155 (1968)), and in certain of the Aspergilli e.g. A. niger and A. foetidus (my copending patent application No. 13,826/70), now Pat. No. 3,743,503. Preferably also the component (a) or (b) should be capable of inducing interferon production in live mammals. (This can be confirmed by the method of Lampson et al. G. P. Lampson, A. A. Tytell, A. K. Field, M. M. Nemes and M. R. Hillerman Proc. Nat. Acad. Sci., 58 (1967), 782.)

. The antiviral substance of this invention has been described as a principally ionic complex. The complex is characterised by a strong electrostatic interaction between the polymeric cationic moiety and the ribonucleic acid anionic moiety. However, other types of interaction may well operate. For example, it is believed that some form of hydrophobic bonding exists between the two components, although the precise nature of such bonding is not yet understood.

The preferred complexes of this invention are those in which all or almost all of the anionic sites on the doublestranded ribonucleic acid anions are neutralized by the quaternary cationic sites on the quaternary polymer. Such complexes may be termed 1:1 complexes or highly neutralized complexes.

' The complexes of this invention may be prepared by a process which comprises contacting, in solution, organic polymer cations which have a plurality of quaternary nitrogen sites located at intervals along the polymer chains with either (a) double-stranded ribonucleic acid polyanions, said double-stranded ribonucleic acid being of natural origin or (b) polyanions of a double-stranded derivative of a double-stranded ribonucleic acid of natural origin.

Although all of the complexes of this invention can be made by the above-defined process, the physical characteristics of the complexes depend to some extent on the detailed method by which the polycations and polyanions are brought into contact. For the purposes of explanation the complexes of this invention can conveniently be divided into two arbitrary classes, namely those which are soluble in 0.15M NaCl solution (hereafter referred to as. soluble complexes and those which are insoluble in 0.15M NaCl (hereafter referred toas insoluble complexes).

In general, we have observed insoluble complexes are prepared by slowly adding a solution of the polyquaternary component to a dilute solution of the double-stranded ribonucleic acid component (e.g. about 0.5 mg./ml.). For example, a solution of an organic polymer containing the desired polycation can be made up in an aqueous solution of an inorganic salt, e.g. NaCl. A similar solution of the ribonucleic acid component can be made up in an aqueous solution containing an inorganic salt. The polyquaternary solution may then be added to the ribonucleic acid solution with stirring and, providing the molarity of the resultant salt solution is not too high, the desired complex will precipitate directly. If it does not precipitate, the solution can be diluted to reduce the molarity below the critical level and the desired complex will then precipitate. Alternatively, a physical mixture of a neutral polymer containing the polycation and the ribonucleic acid or ribonucleic acid derivative can be added to a salt solution, and if necessary, the resultant solution can be diluted to precipitate the desired complex. Preferably, in both of the above methods of preparing insoluble complexes, a molar excess of the organic polymer polycation is contacted with the ribonucleic acid polyanion (the molar excess being calculated on the basis of the number of basic sites capable of reacting with the phosphoric acid sites on the ribonucleic acid polyanions).

It will be realized from the above paragraph that it is relatively easy to produce the insoluble complexes of this invention. More care, however, is required to produce the soluble complexes.

To produce the soluble complexes a solution of the polyquaternary compound is added slowly, with stirring, to a solution of the double-stranded ribonucleic acid or ribonucleic acid derivative in aqueous NaCl, 0.15M, until just before precipitation begins or until only a small amount of precipitation takes place. Any precipitate is then removed, leaving the desired complex in solution. In general, extensive precipitation should be avoided, since homogeneity of the complex left in solution cannot be guaranteed if much precipitation is allowed to take place. I prefer to add the solution of polyquaternary compound to a solution of the double-stranded ribonucleic acid or ribonucleic acid derivative containing not less than about 5 mg./ml. (e.g. 5-20 mg./ml.) since at higher dilutions it appears that almost all the ribonucleic acid is precipitated as insoluble complex.

As has already been indicated, the preferred complexes of this invention are those having a high degree e.g. more than 60%, preferably more than 75%) of charge neutralization. Also, because they are more conveniently administered and in some cases possess advantageous biological properties relative to the insoluble complexes, those: complexes which are soluble in isotonic saline are pre ferred. However, soluble complexes cannot conveniently be prepared using all polyquaternary compounds, since with some, precipitation of an insoluble complex occurs before suflicient polyquaternary compound has been added to achieve a high degree of charge neutralization. While it must ultimately be a matter of trial and error to test whether a soluble complex can be: made. w h y particular polyquaternary compound, certain guidelines can be laid down. Thus, it has been noted that when polyquaternary compounds of structure (I) are employed, there is a direct relationship between the equivalent weight of the polyquaternary compound and the degree of neutralization obtainable with that polyquaternary compound before precipitation of insoluble complex begins. (The equivalent weight is the molecular weight of the polycation divided by the number of basic nitrogen sites per molecule.) It has been noted that with quaternary polycations of structure (I) having a low equivalent weight, more polycation can be complexed with the ribonucleic acid before precipitation of insoluble complex begins than with quaternary polycations of structure (I) having a higher equivalent weight. This tendency will be illustrated in the examples presented later in this specification. It 'is perhaps worthwhile emphasizing again that by insoluble complexes of this invention, is meant complexes which are insoluble in 0.15M NaCl solution. In fact most, if not all, of these insoluble complexes can be dissolved in concentrated electrolyte solutions, but in such concentrated electrolyte, the complexes are dissociated. This is in contrast with the soluble complexes which, when in solution in 0.15M NaCl, are believed to be substantially non-dissociated.

The complexes of this invention (both soluble and insoluble) are antiviral in activity, having a wide spectrum of activity against a variety of DNA and RNA viruses, e.g. encepholomyocarditis (EMC) virus, Semliki Forest virus, Foot and Mouth disease virus and Herpes Simplex virus. It is believed that their mode of action is principally by induction of interferon in host cells, thereby conferring protection against virus attack. For this reason it is believed their primary-utility lies in the prophylaxis of virus infection rather than in the treatment of established infections. The complexes are in general more resistant to ribonuclease degradation than the double-stranded ribonucleic acid itself.

Thus, in another of its aspects, the present invention provides a pharmaceutical composition comprising an antiviral complex as defined hereinbefore and one or more pharmaceutictlly acceptable carriers.

The choice of pharmaceutical carrier is determined by the preferred mode of administration and standard pharmaceutical practice. The mode of administration may be by injection, e.g.. subcutaneously, intravenously or intramuscularly, in which case the carrier will be an injectable liquid in which the complex may be dissolved, or suspended as a fine dispersion. However, even with the soluble complexes of this invention it may be difficult to redissolve them once they have been isolated (e.g. by freeze-drying) and we therefore prefer to form the complex in situ in the injectable liquid (e.g. isotonic saline). For topical application the carrier may be a liquid for application to the muscous membranes. The composition of Compounds 1-7 were prepared by refluxing the appropriate N,N,N,N-tetramethyldiamine (0.1 mole) and dibromoalkane (0.1 mole) in methanol (300 ml.) for 5 hours. The solution was cooled and concentrated at reduced pressure. The residue was dissolved in water (250 ml.) and the solution extracted with ethyl acetate (3X 150 ml.). The aqueous solution was then concentrated at reduced pressure to half volume and dialysed against water (3X 5 lit.). The solution inside the dialysis bag was then concentrated at reduced pressure and on trituration of the residue with ethanol (ca., 100 ml.) a White solid was obtained.

For the preparation of compound 8, 0.09 mole of N,

N,N',N tetramethyl propane 1,3 diamine and 0.08

mole of 1,4 dibromobutane were used, and for the preparation of compound 9, 0.1 mole of N,N,N,N' tetramethyl butane 1,4 diamine and 0.08 mole of 1,4 di bromobutane were used.

For the preparation of compound 10,0.1 mole of N, N,N',N' tetramethyl propane 1,3 diamine and 0.08 mole of 1,4 dibromobutane were used and the dialysis stage was omitted. The residue obtained from concentration at reduced pressure of the ethyl acetate-extracted aqueous solution was triturated three times with boiling isopropanol (3 X 500 ml.), filtered and dried. The product was obtained as a fine white powder.

Compound 11 (hexadimethrine bromide) was purchased as Polybrene from Aldrich Chemical Company.

(ii) Characterisation of Polyquaternary Ammonium Starting Materials In their paper on the macromolecular properties of hexadimethrine bromide, Barlow et al., Proc. Soc. Exp. Biol. Med., 113, 884 (1963), suggest that reactions which could terminate the polymerization are cyclisation and dehydrobromination.

The NMR spectra (determined in D 0) of hexadimethrine bromide and the other saturated polyquaternary ammonium compounds synthesized in the present work provide no evidence for the presence of vinylic protons which would be present if chain termination occurred as a result of dehydro bromination. There is, however,

this invention may be administered alone or in combina- (i) Preparation of Polyquaternary Ammonium Compounds as Starting Materials Table l lists the physical properties of a number of polyquaternary ammonium compounds which were syn thesised as follows:

in each case a singlet at 6:2.2-3 pm. which can be assigned .to -N(CH protons. The NMR evidence therefore suggests that the polymer chains terminate in dimethylamine groups. Assuming this to be correct, it

- is possible to calculate an average molecular weight for the polymers from the ratio of the intensites of the resonances assigned to protons (chain propagation) and those assigned to N('CH3) protons '(chain termination). Unfortunately the bands cannot be integrated with accuracy because of overlap of other resonances, but since the bands are both sharp singlets, the relative peak heights are taken as a first approximation, e.g.: For hexadimethrine bromide, Compound 11, a=6; b=3

Intensity p .In Table 1 structural and analytical data obtained for the polyquaternary compounds is summarized. Most of the polymers are extremely hygroscopic and as a consequence accurate elemental analyses are diflicult to obtain.

TABLE 1.POLYQUATERNARY AMMONIUM COMPOUNDS Nmr data (D20) Total Elemental analysis, percent N(CH;) 2- number Equiv.

I N(OHa)2 quater- Weigh Calculated Found nary (.MW.) Yield, 8 Peak 5 Peak sites Compound b percent 0 H N Br 0 H N Br (p.p.m.) height (p.p.m.) height (2x) M.W. (2x+2 3 3 12. 7 38. 0 7. 7 9. 3 45. 0 36. 9 7. 8. 7 44. 1 3. 32 22. 6 2. 28 3. 95 11. 4 2, 020 2 4 18. 1 36. 5 7. 4 8. 7 47. 4 35. 4 7. 4 8. 3 46. 6 3. 36 24. 8 2. 95 1. 0 49. 6 8, 350 16 3 4 85. 5 38. 5 7. 7 8. 3 45. 5 37. 7 7. 9 7. 9 43. 6 3. 23 20. 95 2. 97 0. 8 52. 4 9, 200 16 3 31. 1 39. 8 7. 3 8. 7 44. 3 37. 0 7. 3 7. 6 42. 2 3. 26 22. 4 2. 2. 7 17. 1 3, 070 16 4 4 9. 5 40. 7 8. 0 8. 1 44. 2 40. 4 8. O 7. 8 42. 0 3. 09 16. 7 2. 47 1. 1 30. 4 5, 620 173. 5 6 4 41. 2 44. 1 8. 5 7. 4 40. 42. 9 8. 4 7. 0 39. 0 3. 12 24. 6 2. 99 1. 0 49. 2 9, 720 19 6 6 50. 5 46. 8 8. 9 7. O 37. 3 45. 7 8. 8 6. 5 36. 2 3. 10 24. 3 2. 89 1. 7 28. 6 6, 130 200 3 4 33. 1 39. 8 8. 0 8. 9 43. 3 38. 8 7. 9 8. 0 42. 3 3. 23 23. 0 2. 30 4. 05 11. 4 2, 100 157 4 4 20. 2 41. 3 8. 2 8. 4 42. 2 40. 3 8. 1 8. 1 42. 8 3. 17 25. O 2. 62 3. 2 15. 6 2, 950 168 3 4 51. 3 40. 5 8. 1 9. 3 42. 1 37. 7 8. 0 8. 3 43. 3 3. 23 24. 6 2. 48 6. 3 7. 8 1, 480 r 151 6 3 42. 8 8. 3 7. 8 41. 1 41. 3 8. 5 7. 4 40. 7 3. 19 20. 0 2. 75 1. 7 23. 6 4, 590 171 I CH2CH=CHCH2 EXAMPLE 2 The nucleic acld thus obtained had identical physical Preparation of an isotonic saline insoluble complex from d.s. RNA isolated from P. chrysogenum virus-like particles and hexadimethrine bromide.To a stirred solution of the d.s. RNA (100 mg.) obtained from the virus particles found in P. chrysogenum ATCC 10002 in 0.15M sodium chloride (200 ml.) at room temperature was added a solution of hexadimethrine bromide (200 mg.) in 0.15M sodium chloride (200 ml.). A precipitate was obtained. The reaction mixture was stirred for 16 hr. at room temperature and then centrifuged. Measurement of the U.V. spectrum of the supernatant solution indicated that it contained no nucleic acid. The precipitate was washed with water (200 m1.) and then with methanol (200 ml.), and dried at room temperature in vacuo giving the product as a pellet (141 mg.).

Properties and Characterisation of the Complex 1. The product is soluble in 0.6M NaCl and higher molarities.

2. Ultraviolet spectral determinations with weighed quantities of the complex indicated that it contained 68i10% nucleic acid. A neutral complex (1 N+ per phosphate) would contain 76% nucleic acid.

The product is probably the 1:1 neutral complex, but incomplete drying as a consequence of tightly bound solcharacteristics (hyperchromicity, T gel electrophoresis pattern) to those of the original d.s. RNA. Moreover, gel permeation chromatography of a solution of the complex in 1.5M Na'Cl on a Bio Gel 150M column gave an identical U.V. trace to that of the original d.s. RNA.

4. Reduction of the molarity of a solution of the complex to below 0.6M in sodium chloride results in precipitation of the complex.

Antiviral Activity of the Complex where N=number of animals in a group vent may have resulted in a low nucleic acid assay. n=number of animals on day x Time Virus challenge between drug and 10 10" subsequent virus Dose Survival Survival Compound (hours) g/mouse) Mortality time Mortality time d.s. RNA-hexadimethrine complex b 72 1 8/ 10 6. 0 8/10 6. 7 10 7/10 7. 0 1/10 50. 0 0/ 10 00 0/10 00 d.s. RNA 0 72 1 10/ 10 4. 7 8/ 10 5. 9 10 7/10 6. 9 6/10 11. 6 100 7/10 8. 8 1/10 50. 0

Untreated 19/20 18/20 Dose refers to amount of d.s. RNA present in each case.

9 Toxicity of The Complex 7 The toxicity of the d.s.-RNA starting material and that of the hexadimethrine-d.s.-RNA complex were compared in 16-20 g. mice of the strain CD1. The compounds were administered by intraperitoneal injection, and the animals observed for 10 days after.

TABLE 3 a Dose refers to d.s. RNA present in each case. b Administered as a solution in 1.5M NaCl. Administered as a solution in 0.15M NaCl.

EXAMPLE 3 Preparation of isotonic saline-insoluble complexes from d.s. RNA isolated from P. chrysogenum virus-like particles and polyquaternary compounds 3, and 10.

The procedure described in Example 2 for the preparation of the isotonic saline-insoluble complex of hexadimethrine bromide and d.s. RNA was followed exactly using polyquaternary compounds 3, 5 and described in Example 1. The resultant complexes were designated C C and C respectively. Complex C was only soluble in aqueous NaCl at ionic concentrations higher than 1.5M; complex C was soluble in aqueous saline at ionic concentrations greater than 0.6M; while complex C was soluble in aqueous NaCl at ionic concentrations greater than 0.45M. Thus, there appears to be a relationship between 10 To a solution of d.s. RNA (154 mg.) in 0.15M NaCl (10 ml.) a solution of the polyquaternary ammonium compound 1 (a=b-=3, x=5.7; 67.5 mg.) in 0.15M NaCl (5.4 ml.) is added in small portions with constant agitation. A clear solution is obtained which can be diluted with 0.15M sodium chloride to any desired concentration.

TABLE 5.POLYQUATERNARY AMMONIUM COMPOUND DOUBLE STRANDED RNA COMPLEXES [A solution of the appropriate polyquaternary ammonium compound in 0.15M sodium chloride (5.4 ml.) was added in small portions, with constant agitation, to a solution of double stranded RNA (154 mg.) in 0.15M sodium chloride (10 ml.)

Weight polyquaternary, Electro Polyquaammonium phoretic ternary compound Percent mobility ammoiuum, added without phosphate electro- Comcompound precipicharge phoretic glex structure tation neutralmobillity o. (a/b (mg.) isation of d.s. RNA

01 3/3 67.5 100 0.53 C2 2/4 57. 9 80 0. 62 C3 3/4 14 60.5 80 0.62 04 butene/8"- 57.4 80 0.66 05 4/4 46.5 60 C6 61/4 33. 9 40 0. 75 C7 fi/GN-fl 35. 8 40 0. 79 C8 3 /4 -4 70. 1 100 0. 58 C9 4/4 -u 60.0 80 0.56 010"... 3/4 67. 5 100 0. 56 C11 6/3 45. 9 60 0. 73

=See Table 1. Based on equivalent weights from Table 1 and average M.W. d.s. RNA nucleotide=844.5.

"For electrophoresis 4% polyacrylamide gels containing 0.04% bis acrylamide were prepared in glass tubes, 4 mm. internal diameter Running bufier was tris (0.04M). sodium acetate (0.2M), EDTA (0.002M), pH 7.8. Electrophoresis was carried out at 5 mA. [tube for 2 hr. d.s. RNA. is separated into 3 bands with only slightly different mobilities. The relative mobility values [or the complexes are based upon the median mobility of the 3 bands.

Did not move onto gel.

the molecular weight of the polyquaternary compound and the ionic concentration required for dissociation of Ph h 1 f 1 its complex with dsi RNA. ysico-c em1ca Properties 0 Comp exes The antiviral activity and toxicity of complexes C C 1. When dilute solutions of the complexes in 0.15M and C were tested as in Example 2. The results are sumsodium chloride are heated to about 50", precipitation marized in Table 4. occurs.

TABLE 4 Number dead/total number in group Antiviral data (EMC) Compound administered 3 days prior to virus infection Compound administered 1 day prior to virus infection Virus dose, 10- Virus dose, 10-

Virus dose, 10- Virus dose, 10*

Toxicity (i.p.)

Compound dose (mg/ g) Complex number 5 0. 5 0. 05 5 0. 5 0. 05 5 0. 5 0. 05 5 O. 5 0. 05 200 160 100 80 40 25 20 (mg/kg.)

O/lO 10/10 4/10 5/10 10/10 2/10 5/10 9/10 0/10 25 Dose refers to amount of d.s. RNA present in each case. d.s.

EXAMPLE 4 Preparation of isotonic saline-soluble complexes of polyquaternary compounds and d.s. RNA isolated from the virus-like particlesfound in P. chrysogenum.

Complexing procedure RNA administered as a. solution in 0.15M N aOl and complexes as a solution in 1.5M

b At every dose level deaths occurred 2 days later than with d.s. RNA alone.

2. Electrophoresis.4% polyacrylamide gels containing 0.04% bisacrylamide were prepared in glass tubes, 4 mm. internal diameter. Running buffer was tris (0.04M), sodium acetate (0.02M), EDTA (0.002-M), pH 7.8. Electrophoresis was carried out at 5 ma./tube for 1-3 hr.

The complexes generally moved as discreet single bands with a lower mobility than d.s. RNA. Within the series, the electrophoretic mobility of the complexes decreases with increasing neutralization of the nucleic acid phosphate charge (Table 5). Complex C5 gave a highly aggregated gel rather than a viscous solution, and on electrophoresis did not move on to the polyacrylamide gel.

3. Degradation by Ribonucleases of Human Serum.- Three representative complexes were tested for their relative susceptibility to dilute human serum.

11 12 The incubation conditions were: 2. Antiviral Activity.The protection afforded by the I complexes against mouse EMC virus is summarized in igg i g i g'g i g g Table 7 and Table 7A gives the results found with un- 0.1 ml. human serum diluted with water 1 in to 1 in 100. treated controls 5 Complexes C1, C2, C3, C4, C9, C and C11 have The mixture was incubated for min. at 37 C., after better antiviral activity than d.s. RNA when given 3 days which 0.1 ml. was removed, mixed with 0.1 ml. buffer D before virus infection, and as good or better when given (0.05M NaCl, 0.001M EDTA, 20% sucrose, pH 7) and 1 day before infection.

TABLE 6.-TOXICITY OF COMPLEXES [The acute toxicity was determined for mice, strain CD1, weighing 18-22 g. The animals were observed for 7 days after dosing by the intraperitoneal route and deaths recorded] Number dead/total number in group at stated dose (mg/kg) Approximate LDw 200 100 80 50 25 20 12. 5 10 5 2. 5 (mg/kg.)

Complex number:

n The dose refers to the nucleic acid component in each case.

TABLE 7.ANTIVIRAL ACTIVITY OF COMPLEXES [Mice, strain CD1, weighing 18-22 g., were administered compounds by the intraperltoneal route either 24 or 72 hours prior to infection with EMC virus, also by the intraperitoneal route. Deaths were recorded daily for 13 days] Number dead total/number in group Animals dosed 3 days prior to Virus infection Animals dosed 1 day prior to Virus infection Virus dose 10' Virus dose 10- Virus dose 10- Virus dose 10 Compound dose (mg/kg.) Experiment Complex number number 5 0.5 0. 05 5 0. 5 0. 05 5 0. 5 0. 05 5 0. 5 0. 05 1 02 4/10 9/10 10/10 0/10 4/10 7/10 2/10 0/10 2/10 0/10 0/10 1/10 03 3/10 6/10 10 2/10 5/10 6/10 0/10 0/10 7/10 0/10 0/10 2/10 011 5/10 10/10 10/10 0/9 7/10 9/10 2/10 3/10 7/10 0/8 0/10 3/10 d.s. RNA 8/10 10/10 10/10 5/10 5/10 8/10 1/10 1/10 5/9 O/10 1/10 3/10 2 04 0/10 8/10 8/10 1/10 2/10 7/10 0/9 1/10 2/10 0/10 4/10 1/10 C5 2/10 7/10 10/10 1/10 10 9/10 2/10 5/9 8 10 1/10 3/10 4/10 C6 3/10 8/10 10/10 1/9 7/10 8/10 0/10 4/10 5/10 0/10 0/9 3/10 d.s. RNA. 3/7 8/10 8/10 0/10 4/10 7/10 0/10 2/10 0/10 1/10 1/10 2/10 3 010 1/10 7/10 9/10 1/10 7/10 9/10 2/9 2/10 5/10 0/10 1/10 8/10 d.s. RNA 9/10 10/10 10/10 4/10 5/10 10/10 2/10 5/10 9/10 0/10 4/10 6/10 4 /1 1 /1 /10 1/10 7/10 0/10 0/10 6/10 1/10 1/10 0/10 8/10 10/10 1/10 4/10 5/10 2/10 4/10 9/10 O/10 1/10 1/10 09 1/10 8/10 1/10 3/10 0/10 1/10 1/10 d.s. RNA 5/10 10/10 10/10 5/10 6/10 4/10 2/10 4/10 5/10 0/10 0/10 1/10 *The dose referes to the nucleic acid component in each case.

-100 ,ul. subjected to electrophoresis for 3-4 hours TABLE 7(A).UNTREATED CONTROLS (electrophoresis condltrons as in 2 above).

The gels were scanned using an ultraviolet spectrometer 50 Number dead/Mal number in group equipped with a linear transport scanner, and the peaks Virus dose 10- Virus dose 10- integrated. Experiment number:

1 20/20 19/20 Percent degradation of nucleic acid H Serum dilution 01 cs (:10 d.s. RNA 20/20 o 3 35 g 1? 3g EXAMPLE 5 o 11 100 Based on disappearance of original nucleic acid band. Further Polyquaternary Ammonium Salt Thus complexes C1 and C10 are considerably more (Compound 12) resistant to human serum ribonucleases than is d.s. RNA I itself and C3 is completely resistant. Compound 12 W Prepared from 1151,4633

methylbutane-1,3-d1am1ne (0.1 mole) and 1,4-d1bromo- 13101081631 Propertles of Complexes butane (0.1 mole). After dialysis and concentration un- 1. Toxicity.--Acute intraperitoneal toxicities were dereduced P 1 a Yesldue Was formed Whlch Was termined in mice (Table 6). The complexes were generally mturated Wlth lsopropanol as for Compound slightly more toxic than d.s. RNA. The minimum lethal CH3 dose for d.s. RNA is about 20 mg./kg., whereas for many 6B of the complexes it is 1012.5 mg./kg. The highly aggre- (CH3)2N ((EHCHZCHB) IYI(CH2)4 gated CS was considerably less toxic, however, and the CH3 CH3 complexes C6 and C7 prepared from the more hydrophobic polyquaternary ammonium compounds and in CHzCHZCH N(CHa)2 which there was less neutralization of the phosphate charge, were also slightly less toxic than d.s. RNA. CH3 CH3 13 14 Compound 12 (an isomer) EXAMPLE 6 (a) Preparation of an isotonic soluble complex from Elemental Analysls-calculated Percent: C, N-oxidized d.s. RNA derived from P. chrysogenum 8.2; N, 8.4; Br, 42.0. Found percent: C, 40.8; H, 8.2; N,

74 Br 411 The N-oxidized d.s. RNA was prepared (using the 5 d I n s Nmrdata (D20): N (CH3)T 5:325 pp 5 fpglolgewsure outllned 1n British Patent 1,284,150), as

1 2 s)2 P99k D.s. RNA (300 mg.) in 0.04M potassium acetate, pH

- qulva em 1 8.2 (300' ml.) was treated with a solution of m-chloroperbenzoic acid (7.5 g.) in ethanol (150 ml.) and the Pfepal'atloll all lsotomc $311116 111501111316 Q pl solution kept at for 1 hour. On precipitation with fro L8. RN Isolated from chl'ysogemlm vll'lls'llke ethanol (900 ml.) the oxidized d.s. RNA was separated Partlcles, and Polyquatefnary Compound 12 by centrifugation, washed with ethanol (2X 500 ml.) and dissolved in 0.15M NaCl (15 ml.) to give a solution of The procedure described in Example 2 was followed, N- idi d 1, RNA (15.9 mg./ml,) using the polyquaternary compound described above. The 15 complex, Complex C was soluble in M sodium chloride A M solution. 260

The antiviral activity and toxicity of this insoluble comratio for the product was 0.60 compared with 0.45 for plex is given in Table 8. d.s. RNA.

TABLE 8 [Antiviral activity and toxicity of d.s. RNA and insoluble and soluble d.s. RNA-polyquaternary ammonium complexes 0.; and 012 Number dead out of 10 in each group Antiviral data (EMC) Virus dose 10' Virus dose 10- Virus dose 10' Virus dose 10- Toxicity (i.p.)

Compound dose (mg/kg.)

LDso 0. O5 100 50 12. 5 (mg/kg.)

Compound 5 0.5 0.05 5 0.5 0.05 5 0.5 0.05 5 0.5

d.s. RNA 7 9 10 3 6 10 0 4 9 0 2 1 9 5 1 25 Complex G12 3 6 10 1 2 4 1 1 4 0 0 0 10 10 10 25 Complex 03 10 10 8 1 2 2 0 3 5 0 0 0 2 0 0 100 Nora-See the following table:

Untreated controls Virus dose 10 Virus dose 10 Mortality 20/20 19/20 (0) Preparation of an isotonic saline soluble complex To a Solution of this dsi RNA N oxide (636 mg) in RNA Isolates; from chrysogenum and poly- 40 0.15M NaCl (4 ml.) was added dropwise with constant quaternary Compoun 12 stirring a solution of compound 10 (22.3 mg.) a 0.15M

The soluble C0mplexC0mplex 12was prepared NaCl (2.36 ml.) (equivalent to 80% neutralization). A from d.s. RNA (217 mg.) in 0.15M NaCl (10 ml.) and small amount of precipitation occurred. The precipitate a solution of polyquaternary compound 12 (84 mg.) in was removed by centrifugation and discarded. The super- 0.15M NaCl (11.7 ml.) in the manner described in Examnatant indicated a nucleic acid complex concentration of ple 4. The complex has 80% charge neutralization, and 9.3 mg./ml. As for the parent N-oxidized d.s. RNA on has a mobility 0.44 times the median mobility of d.s. electrophoresis this polyquaternary ammonium complex RNA. did not move into the gel.

The antiviral activity and toxicity of this complex are The antiviral activity and toxicity of this complex, given in Table 8. Complex C13, are recorded in Table 9.

TABLE 9 [Antiviral activity and toxicity of d.s. RNA and soluble modified d.s. RNA-polyquaternary ammonium complexes C13, C14 and 015] Number dead out of 10 in each group Antiviral data (EMC) Virus dose 10- Virus dose 10- Virus dose 10- Virus dose 10- Toxicity (i.p.)

Compound dose (mg/kg.)

Experiment Compound 5 0.5 0.05 5 0.5 0.05 5 0.5 0.05 5 0.5 0.05 1 d.s. RNA 10 9 10 5 9 o o 4 s 0 o 3 d.s. RNA/N-oxide- 9 10 9 8 s 8 9 9 1o 0 5 4 Complex 013 9 10 10 7 8 9 8 1o 10 0 4 5 2 d.s. RNA 9 10 10 3 5 s 1 5 9 1 1 3 d.s. RNA/C1120 10 9 1o 2 2 6 7 9 10 0 2 6 Complex 014. l0 9 9 6 10 6 7 10 10 0 1 6 Complex 015 1o 10 10 9 10 7 5 8 9 2 3 5 B 5 mice per group for toxicity test. b 6 mice only in the group. EXAMPLE 7 No'rE.Sec the following table: Preparation of an isotonic saline soluble complex from formaldehyde modified d.s. RNA derived from P. UNDOSED CONTROLS, MORTALITY chrysogenum virus particles Virus dose 10- Virus dose 10- Formaldehyde modlfied d.s. RNA was prepared as Experiment f ll 1 19 20 17 20 2 1920 19720 Potassium acetate (0.5 g.) was added to a solution of d.s. RNA (500 mg.) in 0.15M saline (25 ml.) to give 15 a 0.2M acetate solution pH 8.6. Formaldehyde solution (13 ml., ca. 100 fold excess) was added and the pH adjusted from 6.7 to 8.0 with dilute sodium hydroxide. The reaction solution was incubated at 60 C. for 0.5 hr., cooled to room temperature, dialysed vs 0.15M saline 16 EXAMPLE 9 (a) Preparation of the isotonic saline soluble complexes from the d.s. RNA isolated from P. stoloniferum viruslike particles gg gi ii s 3 2 solutlon contammg The d.s. RNA (4 ml. at 20 mg./ml. isolated from P.

The following physical characteristics were measured: stolomfemm VLPS Klemschmldt Nature Tm (1/10 SS0.) 86 c. C. for d.s- RNA). 1968, 220, 167 and G. T. Bankset al., Nature, 1968, 218,

R 78 94 C 84 20 542) in 0.15 ml. NaCl was mixed dropwise W1th com- Me mg ange 9 pound 10 (33.8 mg. m 2.7 ml. 0.15 M NaCl) by the Hc 37% usual procedure. After the addition of 2.2 ml. of this PAAGE 3 Peaks to N Solution, no more was added as there were signs of rebut Wlth lower, moblhty cipitation. The Complex C18 so formed thus contained and less dlstmct' 85% neutralization. PAGE(1nfrmam1de) Moblhty 55% of 15 Similarly Complex C19 Was prepared from P. stoloni- TO a Solution of the formaldehyde modified RNA ferum d.s. RNA and compound 3. 66% neutralization (96 mg.) above in 0.l5M NaCl (10 ml.) a solution of Qurred Compound 10 in0-15M Nacl q Electrophoretic mobility relative alent to 100% neutralization) was added dropwise with c l to stolonifemm (18 RN 1 constant agitation- 20 P. stoloniferum d.s. RNA 1 and 1 The complex, Complex C14, had a mobility of 0.33 Complex 3 Q8 and 0 83 relative to d.s. RNA and 0.44 relative to formaldehyde Complex 19 078 and O '78 modified d.s. RNA.

Com lex C15 was re ared from the same formaldemajor peaks e Obtained r jferum d.s. hyde g i using compound 3 (37] mg.) gill; fitllgez' relative mobility for the slower moving peak is in 0.15M NaCl (equivalent to 86% neutralization). some precipitate formed which was removed by (b) Preparation of an isotonic saline insoluble complex trifugatieh- The Supernatant Contained the COmPIeX C15 from the d.s. RNA isolated from P. stoloniferum virus- With a eleetlophoretle mobility of like particles and Hexadimethrine Bromide relative to d.s. RNA and 0.66 relative to formaldehyde modlfied P. stoloniferum; d.s. RNA (4 ml. at 20 mg./ml.) in

The ant1v1ral act1v1t1es and toxicltles of complexes C14 015M Nacl was diluted to 160 with 0.15M Nacl and C15 and hsted m Table and a solution of hexadimethrine bromide (160* mg.) in

EXAMPLE 8 0.15 M sodium chloride (160 ml.) was added at room Preparation of the isotonic saline soluble complexes from temperature A p t p ig e l-t After 18 t the low molecular wei ht d.s. RNA derived from at TOOIH p r e Pfeelplete was 60 e0 e V P. chrysogenum g icientlrlifujaticin,dvvashegilvlwlighcfvater and methanol, and

The low molecular wei ht d.s. RNA 104.5 m isoy 1550 Y m lated from chrysogemmf (R COX,Kanag2;g1i21gam 40 The material had properties s1m1lar to those outlined and E. s. Sutherland, Biochem. J. 1970, 120, 549, and i fgf g z g fg fii zg g i i s 5 53 Biochem. J., 7 125, 655. in 0.15M NaCl 5 ml. was treated \ili th compound 10 (45.8 mg.) in 0.l5lV I mated NaCl (5.45 ml.) for 100% neutralization as described EXAMPLE in Example 4 to yield Complex C16.

N B I h preceding examples, wherever a ref Preparation of an isotonic saline soluble and insoluble to d RNA f o Chrysogenum. occurs, i i to be complex from the Repllcative Intermed1ate of the sus-3 taken as meaning the high molecular weight fraction demutant of f2 cohphage scribed by Cox et al. in the above reference.

Similarly on treatment with polyquaternary ammonium To a solution of this RNA (100 mg.) in 0.15M NaCl compound 3, (41.0 mg.) in 0.15 M NaCl (4.36 ml.) for (5 ml.) was added with constant agitation a solution of neutralization, the low molecular weight d.s. RNA compound 10 (43.8 mg.) in 0.15M NaCl (5 ml.). No from P. chrysogenum formed the soluble derivative, obvious precipitation occurred on addition, but on com- Compound C17. pletion a floculent precipitate settled which was separated Relative to the low molecular weight d.s. RNA com- 55 by centrifugation. The supernatant (RNA concentration plexes C16 and C17 had mobilities of 0.55. 5 mg./ml.) was designated Complex C20. The residue The antiviral activities and toxicities are recorded in was dissolved in 0.75M NaCl (5 ml.) to give an RNA Table 10. concentration of 9.2 mg./ml. This is Complex Cf.

TABLE 10 [Antiviral activity of d.s. RNA low molecular weight, d.s. RNA (from P. chrysogenum) and low molecular weight d.s. RNA polyquaternary ammonium complexes C16 and 017] Compound administered 3 days prior to virus infection Virus dose 10- Virus dose 10- Virus dose l0 Compound administered 1 day prior to virus infection Virus dose 10* Toxicity (i.p.)

Compound dose (mg/kg.)

Dso Compound 5 0. 5 0. 05 5 0. 5 0. 05 5 0. 5 0. 05 5 0. 5 0. 05 (mg./kg.) (1.8. RNA 6 10 10 1 5 8 6 4 7 0 2 4 44 Low. mol. Wt., d.s. RNA. 9 9 10 4 8 10 6 9 9 3 4 6 10 25 Complex 016 9 10 10 8 9 9 5 6 10 0 1 5 9 19 Complex O17 9 9 1O 3 10 8 4 6 8 1 0 1 10 15 N0rE.See the following table:

Undosed controls Viral dose 10- Viral dose 10- Mortality 19/20 19/20 The electrophoretic mobility of complex C20 was 0.55 of the parent RNA.

The antiviral and toxicity data are given in Table 11.

EXAMPLE 11 18 EXAMPLE 12 Serum Interferon Levels The serum interferon levels for a number of complexes are given in Table 14. Degradatlon by Pancreatlc Rlbonuclease Groups of 6 mice were dosed by the intraperitoneal The rate of degradation of various soluble complexes by route at g. compound (in terms "of RNA) /mouse. pancreatic ribonuclease was compared with that of d.s. Blood was collected by cardiac puncture and pooled. The RNA and the various parent nucleic acids as indicated in interferon levels were assayed by measurement of the T bl 12 d 13 10 reduction in the number of viral plagues, in L-929. Ribonuclease (10,11. of a 1 mg./ml. solution) in 0.15M (mouse fibroblast) cells challenged with EMC virus, sodium chloride was added to a solution 100 ,ug. of the nucaused by pretreatment of the cells with dilutions of sera. cleic acid or its soluble complex in 0.15M sodium chloride The PDD I ml. serum is the reciprocal of the serum dil solution (2.5 ml.), pH 7-7.5. The optical density at 260 tion which reduces the number of plagues to 5.0% of the mm. was then determined at various time intervals. 5 control.

All complexes show increased resistance to pancreatic The soluble complexes show longer duration of high ribonuclease. serum interferon levels.

TABLE 11 [Antiviral activity and toxicity of d.s. RNA, mutant phage d.s. RNA, and soluble and insoluble mutant phage d.s. RNA-polyquaternary ammonium complexes C and Cf] Number dead out of 10 in each group Antiviral activity (EMC) Compound administered 1 day prior to virus infection Virus dose 10- Virus dose 10- Virus dose 10- Virus dose 10" Toxicity (i.p.)

Compound dose (mg./kg.)

LD Compound 5 0.5 0. 05 5 0. 5 0.05 5 0. 5 0.05 5 0 5 0.05 100 50 12. 5 (mg/kg.)

d.s. RNA 7 10 10 2 3 8 3 3 10 0 0 2 7 44 Mutant phage d.s. RNA.- 10 9 10 8 9 6 10 9 9 2 3 3 10 10 10 Complex C20 8 9 10 7 9 7 8 10 8 4 4 2 10 10 22. 5 Complex Cf 8 9 10 6 10 10 5 9 10 0 3 4 10 9 10 NOTE .See the following table:

Undosed eontro1s Viral dose 10' Viral dose 10- Mortality 20/20 20/20 TABLE 12 [Ribonuclease sensitivity of d.s. RNA and complexes C3 and C10] EXAMPLE 13 Time after addition of ribonuclease Antitumour Activity Expt. 15 N 0. Compound min. min. 1 hr. 2 hr. 5 hr. 18 hr. x He (percent) Mice, stram DBAZ/I, at least eight weeks old, were used and the L5178Y tumour passed weekly in an ascitic d S RNA 6 25 8 2 2 form, 10 cells being injected into the peritoneal cavity.

Complex 0 0 0 0 0 6 Comm 0 0 0 0 0 0 50 About 10 tumour cells in 0.1 ml. PBS were administered on the shaved flank, and the animals dosed at 8, 11, and

TABLE 13 [Ribonuelease sensitivity of d.s. RNAs and their polyquateruary ammonium complexes] Time after addition of ribonuelease j g i 5 min. 10 min. 20 min. 30 min. 1 hr. 2hr. 4 hr. 6 In. 1811!. Experiment number Compound He (percent) 2 d.s. RNA 2. 2 4.3 6.5 10.9 21.7 35.9 47.8 48.9 48. 0

Complex C12 0 0 0 0 0 0 0 O d.s. RNA N-oxide. 13. 1 15.9 20. 5 22. 7 29. 5 34. 1 37. 5 38. 6 40. 9 Complex C13 6. 6 1. 3 2. 5 2. 5 3. 8 6. 3 8. 8 10. 0 15, 0 a A 0. 6 1. s 2. 2 4. 4 11.1 25.6 41. 1 46. 7 4s. 1 d s RNA/CH2O 9. 3 14. 0 19. 3 23. 3 27. 9 33. 7 38. 4 39. 5 39. 5 Complex C14--. 0 0 0 0 1. 3 1. 3 2. 6 5. 0 7. 5 0 0 0 0 1. 3 1. 3 2. 6 3.8 5. 6

4. 1 5. 5 8. 3 12. 5 19. 4 30. 5 45. 8 51. 4 54. 0 6. 3 33. 8 18.8 25. 0 30. 0 37. 5 46. 3 47. 5 47. 5 0 1.5 1.5 4.6 4.6 4.6 6.1 6.1 12.3 Complex C17- 0 0 0 1. 4 1.4 1. 4 1. 4 1. 4 1. 4

5 d.s. RNA" 0 0 0 1.2 6.8 19.3 35. 2 42.0 46.6 P. Stolonife 6. 9 8. 3 11. 1 l3. 9 '21. 1 30. 5 38. 8 41. 6 43. 1 Complex C18 0 0 1. 1 1. 1 '1. 1 2. 2 2. 2 2. 2 2. 2 Complex C19--. 0 1. 2 2. 3 3. 5 3. 5 3. 5 3. 5 3. 5 4. 7

6 d.s. R A 2.4 4.2 8.4 12.0 21.6 39.4 43.4 44.6 48.2 Mut 7. 7 9. 0 12. 8 15. 4 23. 1 33. 3 41. 0 43. 6 46. 1 Complex 020 0 0 0 1.2 1. 2 1. 2 2.5 3.7 5. 0

He percent at minutes.

19- 13 days after. The number of regressions observed per number of animals in a group are recorded in Table 15.

TABLE 14 [Serum interferon levels of various complexes] Time after administration of compound 2 hr. 4 hr. 24 hr. 30 hr. 48 hr.

Serum Interferon PDDso/ml. serum d.s. RNA 330 445 25 0 Comp ex Complex 0 is the d.s. RNA-hexadimethrine bromide insoluble complex.

TABLE [Tumour regression by d.s. RNA and complex 08] Regression after- What we claim is:

1. An antiviral complex which is a principally ionic complex soluble in 0.15 M aqueous sodium chloride solution in which the cations are organic polymer polycations having a plurality of quaternary nitrogen sites located at intervals along the polymer chains, said polycations having the formula wherein each of a and b, independent of the other, is an integer of from 2 to 6 and X is a number which is such that the average molecular weight of the polycation divided by the equivalent weight which is the molecular weight of the polycation divided by the value (2X 2) is not greater than 98, and the anions are either (a) doublestranded ribonucleic acid polyanions, said double-stranded ribonucleic acid being of natural origin, or (b polyanions of a double-stranded ribonucleic acid of natural origin which has been subject to chemical or enzymatic reaction which alters the primary and/ or secondary and/ or tertiary structure, provided that the resultant ribonucleic acid retains a substantial degree of base pairing between complementary strands, said antiviral complex having more than 60% of the anionic sites on the double-stranded ribonucleic acid anions neutralized by the quaternary cationic sites on the quaternary polymer.

2. An antiviral complex according to claim 1 wherein more than of the anionic sites on the double-stranded ribonucleic acid anions are neutralized by the quaternary cationic sites on the quaternary polymer.

3. An antiviral complex according to claim 1 wherein the double-stranded ribonucleic acid component is from the virus particles found in infected strains of Penicillium chrysogenum, Penicillium stolorziferum, Penicillium funiculosum, Penicillium cyaneofulvum, Aspergillus niger or Aspergillus foetz'dus.

4. A process for the production of the antiviral complex of claim 1 which comprises adding slowly with agitation an aqueous electrolyte solution of an organic polymer polycation of the formula wherein each of a and b independent of the other, is an integer of from 2 to 6 and X is a number which is such that the average molecular weight of the polycation divided by the equivalent weight, which is the molecular Weight of the polycation divided by the value (2X+2), is not greater than 98, to a solution of (a) double-stranded ribonucleic acid polyanions, said double-stranded ribonucleic acid being of natural origin or (b) polyanions of a double-stranded ribonucleic acid of natural origin which has been subject to chemical or enzymatic reaction which alters the primary and/ or secondary and/ or tertiary structure, provided that the resultant ribonucleic acid retains a substantial degee of base pairing between complementary strands, in aqueous electrolyte containing not less than about 5 mg./ml. until just before precipitation begins or until only a small amount of precipitation takes place.

5. A process according to claim 4 wherein the aqueous electrolyte solutions are both sodium chloride solutions about 0.15 M with respect to sodium chloride.

6. A process according to claim 4 wherein the solution of double-stranded ribonucleic acid component (a) or (b) contains from 5 to 20 mg./ml. of said component.

7. A process according to claim 4 wherein the doublestranded ribonucleic acid component is from the virus particles found in infected strains of Penz'cillium chrysogenum, Penicillium stoloniferum, Penicillium funiculosum, Penicillium cyaneofulvum, Aspergillus niger or Aspergillus foetidus.

References Cited UNITED STATES PATENTS JOHNNIE R. BROWN, Primary Examiner US. Cl. X.R. 

1. AN ANTIVIRAL COMPLEX WHICH IS A PRINCIPALLY IONIC COMPLEX SOLUBLE IN 0.15 M AQUEOUS SODIUM CHLORIDE SOLUTION IN WHICH THE CATIONS ARE ORGAIC POLYMER POLYCATION HAVING A PLURALITY OF QUATERNARY NITROGEN SITES LOCATED AT INTERVALS ALONG THE POLYMER CHAINS, SAID POLYCATIONS HAVING THE FORMULA 